Calcif. Tiss. Res. 17, 235--248 (1975) 9 by Springer-Verlag 1975
Effects of Chronic Administration of Sodium Diphenylhydantoin ('Dilantin') on Bones and Teeth of the Rat and Hamster: a Preliminary Study P. H. S t a p l e a n d W i l l i a m A. Miller Department of Oral Biology, School of Dentistry, State University of New York, Buffalo, :New York Received July 8, accepted December 8, 1974 Male Charles River rats, 31-days-old, received i.p. injection of sodium diphenylhydantoin (DPH), 100 mg/kg in 0.9% NaC1, once daily for 26-27 days before death. Male Syrian hamsters, 40-days-old, received similar injections of DPH, 25 mg/kg for 46 days, no treatment for 39 days, then DPtt for a further 17 days before sacrifice. All rats receiving DPH gained less weight than the controls, and more than 50% displayed acute neurotexic reactions to the drug; hamsters were not so affected. Morphology and composition of caudal vertebrae, teeth, and jaws from control and DPH-rats were compared on the basis of measurements on radiographs and gross specimens, histological investigation, and determination of % dry volumes of ash, volatile inorganic component, lipid, and organic matrix. DPH-vertebrae were smaller and showed impaired osteogenesis, but chondrogenesis was similar to controls. Overall tail length was similar in both animal groups because caudal intervertebral spaces were wider in DPH-rats, compensating for reduced longitudinal growth of corresponding vertebrae. Incisors were smaller and third molar roots shorter in DPH-rats. In DPH-hamsters the attachment of the periodontal ligament to maxillary incisors was deranged. DPH administration did not change the composition of rat bone or teeth. Densities of dry bones and teeth were in accord with their composition. Possible modes of action of DPH are discussed. Species differences in response of mineralized tissues to DPtt administration are emphasized in relation to reports of rickets and osteomalacia in patients on long term DPH therapy. Key words: Diphenylhydantoin - - Growth - - Skeleton - - Dentition. Introduction Bone f o r m a t i o n a n d calcium m e t a b o l i s m m a y be d i s t u r b e d in p a t i e n t s on long t e r m a n t i c o n v u l s a n t t h e r a p y ( G e n u t h et al., 1972; V a r k e y et al., 1973); b u t w h e t h e r this d i s t u r b a n c e is m e d i a t e d b y a l t e r a t i o n of v i t a m i n D m e t a b o l i s m or n o t is controversial ( K r a i n t z et al., 1973; Villareale et al., 1974). 5,Tone of these r e p o r t s m e n t i o n d e n t a l a b n o r m a l i t i e s , n o r do t h e y suggest t h a t t h e j a w bones m i g h t also be affected. Accordingly, we e x a m i n e d bones a n d t e e t h of r a t s a n d h a m s t e r s p r e s e r v e d from p r e v i o u s e x p e r i m e n t s (Staple et al., 1967 ; Cheng a n d Staple, 1972) for evidence t h a t chronic a d m i n i s t r a t i o n of d i p h e n y l h y d a n t o i n sodium ("Dilant i n " ) affects n o t only t h e skeleton b u t also t h e t e e t h a n d t h e i r s u p p o r t i n g structures. P r e l i m i n a r y a c c o u n t s of this i n v e s t i g a t i o n h a v e a l r e a d y b e e n p r e s e n t e d (Staple a n d Miller, 1973a, b).
For reprints: Dr. P. H. Staple, Dept. of Oral Biology, State University of New York, 4510 Main Street, Buffalo, :New York 14226, U.S.A.
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P . H . Staple a n d W. A. Miller Materials and Methods
Animals, Drug Administration. The handling of male Syrian hamsters (Staple et at., 1967) a n d Charles River rats (Cheng and Staple, 1972) has been described. Essential details of these experiments, which provided bones and teeth for the present investigation, are as follows: Ten 40-day-old hamsters received daily intraperitoneal (tip.) injections of sodium Dilantin (25 mg/kg calculated from weekly individual body weights), for 46 days, no t r e a t m e n t for 39 days, then sodium Dilantin for a further 17 days before death. Ten controls were injected with vehicle. Thirteen 34-day-old Charles River rats received similar injections of sodium Dilantin (100mg/kg calculated from the daily mean body weight), for 26-27 days before death. Thirteen controls received vehicle. Rat Tails and Caudal Vertebrae. Tails from 7 treated and 7 control animals h a d been a m p u t a t e d close to the anal fold a n d stored at --20 ~. Six years later, a t room temperature, they were weighed, measured, and radiographed using 70 kV, 9 mA, a t 75 cm and K o d a k Professional Fine Grain Film. Subsequently, they were immersed overnight in 10% formalin, dried and stored. Caudal vertebrae were dissected out and either subjected to gross compositional analysis on a volumetric basis (Gong et al., 1964; Gong, 1972) or prepared for histologic examination b y routine methods. A second tail radiograph was made to check the location of vertebrae removed. Lengths of selected vertebrae and widths of intervertebral spaces were determined on the initial radiographs viewed 10 x . The microscope was equipped with a filar micrometer eyepiece reading to 0.001 ram. Overall tail length was also estimated b y measuring the length of a thread conformed to the radiographic image of the 4 t h - 2 7 t h (terminal) caudal vertebrae. Rat Teeth and Mandibular Bone. Skulls a n d disartieulated mandibles h a d been stored a t --80 ~ Three years later, they were transferred to modified Newcomer's fixative (Zugibe a n d Fink, 1966) a t --30 ~ for freeze substitution, then stored in 2-propanol a t room temperature. These bones were radiographed to display the dental roots with minimal distortion. After radiography, the t e e t h were extracted after briefly boiling the bones in dilute NH~OH solution or t a p water. Molar root length was determined on radiographs and directly on extracted teeth, b o t h viewed under 10 X, using a stereomicroscope equipped with a n eyepiece disc micrometer. The arbitrary unit employed was approximately 0.1 ram. The dehydrated weight of molar teeth was determined after incubation overnight a t 110% Maxillary third molars were ashed at 800 ~ for 48 h. The composition of an incisor and of mandibular bone (5-15 rag) from t h e angular process from each animal was determined on a volumetric basis as described for caudal vertebrae b u t using a n electrobalance. The basal third of the contralateral mandibular incisor was analysed similarly. Hamster Teeth. Cranial portions containing the maxillary dentition had been defleshed b y Dermestid beetles, washed in dilute ammonia and air dried before storage at room temperature for 10 years. Each preparation was bisected a n d radiographcd. Radiographs were projected a t 6 X onto paper a n d the relative areas of right or left maxillary incisors were determined b y the paper weight method. A series of transverse bands in cementum on the distal surface of the maxillary incisor became visible when the maxilla was suitably oriented a n d viewed b y oblique incident illumination using a steromieroscope a t 10 • Beginning near the ineisal edge, the distance between the first 10 bands was measured with a n eyepiece disc micrometer. The length of the distal root of extracted third maxillary molars was measured, as described for rats. These teeth were t h e n ashed at 650 ~ for 102 h, or a t 800 ~ for 48 h. All data were subjected to a n analysis of variance to determine their significance.
Results
Acute IVeurotoxic Response to Sodium Dilantin F i g u r e 1 s h o w s t h a t a d m i n i s t r a t i o n of 100 m g / k g s o d i u m D i l a n t i n c a u s e d a c u t e n e u r o t o x i c r e a c t i o n s i n a t l e a s t 5 0 % of t h e r a t s . C o n t r o l s b e h a v e d n o r m a l l y .
Diphenylhydantoin Effects on Bones and Teeth
237
260~24O 220i E
Controls
200-t-
180o
rn
Dilantin Sodium
160140120-I00-.:
eo~ 25
50
55
40
45
50
55
60
DAYS
Fig. 1. Male Charles River rats. Above: fractional daily incidence of neurotoxic reactions in treated rats occurring within 20 min of intraperitoneal (i.p,) injection of sodium Dilantin, 100 mg/kg body weight, suspended in 0.9% NaC1. A value of 0.6 indicates that 8 of 13 rats displayed neurotoxicity. Below: days of age and mean body weight of 13 rats receiving sodium Dilantin, 100 mg/kg, as above, and 13 rats receiving saline vehicle, i.p. The arrow indicates commencement of Dilantin or saline injections
O v e r t n e u r o t o x i c i t y was a b s e n t in h a m s t e r s receiving 25 m g / k g s o d i u m Dilantin, b u t these a n i m a l s r e s p o n d e d a b n o r m a l l y to p e n t o b a r b i t a l a n e s t h e s i a (Staple a n d N u t t e r , 1962). A n i m a l Growth
R a t s receiving sodium D i l a n t i n failed to grow at t h e s a m e r a t e as controls (Fig. 1). A t d e a t h a f t e r 26 or 27 d a y s t r e a t m e n t , t h e m e a n b o d y weight of t h e t r e a t e d group was 232.1 :h 5.5 (S.E.M.) g c o m p a r e d with 270.0 :[: 7.1 g for controls ( P < 0.01). I n i t i a l l y , some h a m s t e r s receiving sodium D i l a n t i n failed to gain weight as r a p i d l y as controls. W e i g h t gain of these a n i m a l s d i d n o t a c c e l e r a t e d u r i n g d r u g w i t h d r a w a l n o r level off w h e n D i l a n t i n was r e a d m i n i s t e r e d . W h e n a n i m a l s were sacrificed a t 142 days, t h e m e a n b o d y weight of t h e D i l a n t i n group was 150.6 4- 6.8 g (10) c o m p a r e d with 157.6 ~ 3.3 (10) for controls ( P > 0.05).
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P.H. Staple and W. A. Miller
Table 1. Measurements of lengths on radiographs of caudal vertebrae in stored amputated tails of young adult male Charles River rats receiving sodium Dilantin or vehicle Group of 7 Animals Control (vehicle, i.p.) Sodium Dilantin (100 mg/kg, i.p.)
Mean length (mm) of caudal vertebrate number 5
9
13
17
21
25
Group grand mean length (mm of caudal vertebra
9.065
8.712
8.150
6.929
5.134
3.016
6.834
8.715
8.444
7.872
6.668
5.020
2.960
6.614
Rat Tails and Caudal Vertebrae Length. The mean length of thawed stored tails from 7 treated animals was 15.8• (S.E.M.) cm compared with 16.6~0.22 cm for 7 controls. Corresponding tail weights were 2.6 • 0.13 and 3.3 ~ 0.16 g. These differences (P ~0.05) were found to be spurious following comparison of the tail radiographs with those of previous authors (Baume et al., 1957), which revealed non-uniform tail amputations. Re-determination of tail lengths, measuring between the distal ends of the 4th and 27th (terminal) vertebrate on the radiographs, showed no difference between experimental and control groups (P ~ 0.05). The components of tail length were further investigated by measuring the lengths of vertebrae ~ 5, 9, 13, 17, 21, and 25, and the widths of the corresponding intervertebral spaces, excepting # 25. Table 1 shows that the mean length of selected caudal vertebrae in the Dilantin group was less than in the control group. The complete data also show that in the control group vertebral length progressively declined from # 5-21, whereas in the case of 2 of 7 treated animals vertebra # 9 was longer than # 5. A 3-way analysis of variance (Chilton, 1967) showed that: (1) F = b e t w e e n groups mean square/between animals (within groups) mean square = 1.24 for df 1, 12 ; P ~ 0.05. (2) F = vertebrae • group interaction mean square/animals • vertebrae interaction (within groups) mean square = 1376.6 for df 5, 60; P < 0 . 0 0 1 . This indicates that although the difference between group grand means is not significant, the groups differ significantly when compared on the basis of the mean values for corresponding individual vertebrae in the series. Table 2 shows that: (1) I n both control and treated groups the mean width of caudal intervertebral spaces progressively declined from ~ 4-22. (2) Beginning at intervertebral space ~ 12-13, mean space width in the Dilantin group was greater than in controls. Three-way analysis of variance (a) on the complete data, (b) for data commencing with intervertebral space # 12-13, showed no significant differences between group grand means (P ~ 0.05), nor between means for each intervertebral space considered separately. However, when groups were compared commencing with intervertebral space # 12-13, F = intervertebral space • group interaction mean square/animals • intervertebral space interaction (within groups) mean square = 4.79 for df 5.60; P = 0.001. This indicates a significant difference between the Dilantin and control group intervertebral space widths in the segment of tail comprising intervertebral spaces # 12-22.
Diphenylhydantoin Etfects on Bones and Teeth
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Table 2. Measurements on radiographs of widths of caudal intervertebral spaces in stored amputated tails of young male adult Charles River rats receiving sodium Dilantin or vehicle Group of 7 Animals
Control (vehicle, i.p.)
Mean width (mm) of caudal intervertebral space number 4-5
5 6
8-9
9-10
12-13
13-14
16-17
17-18
20-21
21-22
Group grand mean width of caudal intervertebral space a (a)
(b)
0.664 0.565 0.367 0.327 0.226
0.208
0.198
0.181
0.145
0.126
0.301
0.181
Sodium Dilantin 0.626 0.602 0.360 0.324 0.309 (100 mg/kg, tip.)
0.239
0.228
0.204
0.195
0.173
0.325
0.225
a (a) For whole series # 4 - 5 .... 21-22, (b) # 12-13 .... 21 22 only.
Composition. Caudal v e r t e b r a e # 6 a n d 7 were t a k e n for c o m p o s i t i o n a l analyses. F o r # 6, t h e m e a n d e h y d r a t e d weight was 43.42 ~ 2.50 (S.E.M.) m g for D i l a n t i n compared with 58.14• for control a n d t h e m e a n v o l u m e was 19.66 :~ 1.44 m m a c o m p a r e d w i t h 27.37 • 1.44 m m 3 ( P = 0.01, n = 6) ; corresponding mear~ densities were 2.240 • 0.161 rag/ram a a n d 2.137 =t=0.097 m g / m m a ( P ~ 0.05). F o r # 7 , m e a n d e h y d r a t e d weight a n d volume in t h e D i l a n t i n group were r e s p e c t i v e l y 44.87 • 3.84 m g a n d 20.96 • 2.43 m m 8 c o m p a r e d with 50.04 ~ 2.66 m g a n d 24.95 • 1.10 m m a in t h e control g r o u p ( P > 0.05 for n : 3 ) ; densities were 2.156 ~- 0.068 m g / m m 3 a n d 2.004 =~ 0.026 m g / m m a r e s p e c t i v e l y . On a v o l u m e p e r cent basis, t h e r e were no differences b e t w e e n groups r e s p e c t i n g m e a n values for ash, volatile inorganic fraction, lipid, a n d organic m a t r i x ( P > 0.05). Because of technical difficulties, lipid a n d organic m a t r i x fractions were d e t e r m i n e d o n l y on 4/6 a v a i l a b l e v e r t e b r a e # 6 from each group. Consequently, t h e differences b e t w e e n m e a n values for D i l a n t i n a n d control, r e s p e c t i v e l y 29.2• a n d 34.4:]=6.1 p e r cent, a n d 3 8 . 9 : L 3 . 7 a n d 25.6-]-5.4 p e r cent, failed to r e a c h significance a t t h e 5 % level. Anatomy and Histology. The c o n v o l u t e d contour of tail v e r t e b r a e was m o r e p r o m i n e n t in t h e p r o x i m a l v e r t e b r a e , giving a f l u t e d a p p e a r a n c e along t h e long axis of each bone. T h e fluting is analogous to the t r a n s v e r s e a n d a r t i c u l a r processes of t h e dorsal v e r t e b r a e . T h e r e was also i n t e r n a l buttressing. Because of t h e local h e t e r o g e n e i t y of tissues, g r e a t care was exercised to c o m p a r e c o m p a r a b l e regions. Most of t h e following descriptions a p p l y to t h e midline, where c o n t o u r i n g of t h e a r t i c u l a t i n g surfaces was minimal. A l t h o u g h cellular f i x a t i o n was poor, t h a t of t h e calcified tissues was sufficiently good to give reliable o b s e r v a t i o n s on t h e i r structure. The h i s t o l o g y of v e r t e b r a e # 12-14 showed t h a t t h e j o i n t space was e n l a r g e d in those a n i m a l s which h a d received Dilantin. I n controls, t h e s e c o n d a r y cartilage h a d v i r t u a l l y d i s a p p e a r e d ; however, in t h e e x p e r i m e n t a l a n i m a l s t h e r e was residual s e c o n d a r y cartilage d i s t r i b u t e d i r r e g u l a r l y b o t h c e n t r a l l y a n d p e r i p h e r a l l y a n d t h e d i s t a n c e b e t w e e n joint surface a n d t h e e p i p h y s e a l cartilage was m u c h g r e a t e r (Fig. 2 A a n d B).
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P . H . Staple and W. A. Miller
Fig. 2A and B. Demineralized, longitudinal section of epiphyseal region of vertebra @ 13. Remains of the intervertebral disc are visible (displaced in B). Greater irregularity of calcification is visible in the experimental animal (B) along with greater amounts of residual cartilage, particularly at the joint surface. Haematoxylin and van Gieson, X 40 Fig. 3A and B. Radiographs of tail vertebrae @ 12-14: (A) Control; (B) Dilantin. In B, the wider joint spaces and epiphyseal spaces are clearly visible, as well as interrupted growth lines (arrows). Both vertebrae show longitudinal fluting
Diphenylhydantoin Effects on Bones and Teeth
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E n l a r g e d c o n t a c t r a d i o g r a p h s showed these f e a t u r e s as well as t h e v a r i a t i o n s in v e r t e b r a l size a n d a difference in t r a b e c u l a r coirfiguration (Fig. 3 A a n d B). Some of t h e D i l a n t i n v e r t e b r a e showed i n t e r r u p t e d g r o w t h lines, which could n o t be r e l a t e d to t h e e x p e r i m e n t a l procedures. I n t h e e p i p h y s e a l cartilage t h e p r o l i f e r a t i n g zone was essentially similar in t h e e x p e r i m e n t a l animals a n d controls (Figs. 2 a n d 4 A a n d B), e x c e p t t h a t in some areas c h o n d r o b l a s t clones were more sparse a n d often m o r e irregular in a r r a n g e m e n t in t h e e x p e r i m e n t a l group. I n D i l a n t i n - t r e a t e d animals, t h e h y p e r t r o p h i c a n d calcifying zones were wider with more residual calcified cartilage in a f r e q u e n t l y wider s u b j a c e n t zone of basophilic w o v e n bone. The bone of t h e shaft was t h i n n e r w i t h m o r e irregular lamellae. I n b o t h e x p e r i m e n t a l a n d control a n i m a l s a r e c e n t m a j o r i n t e r n a l r e m o d e l l i n g was evident. L o n g i t u d i n a l sections of v e r t e b r a e # 8 a n d 9, a d j a c e n t to those whose composition was d e t e r m i n e d , showed t h a t t h e y were similar in s t r u c t u r e to t h e m o r e d i s t a l v e r t e b r a e a l t h o u g h t h e i n t e r v e r t e b r a l spaces were" n o t different (Table 2). I n t r a n s v e r s e sections of these v e r t e b r a e , a r o u n d t h e i n t e r n a l buttressing, i n d i v i d u a l t r a b e c u l a e were coarser with larger i n t e r v e n i n g spaces (Fig. 5 A a n d B).
Rat Mandible D i l a n t i n a d m i n i s t r a t i o n d i d n o t a l t e r t h e d r y d e n s i t y nor v o l u m e t r i c composit i o n of bone from t h e m a n d i b u l a r a n g u l a r process.
Rat Teeth The a n g u l a t i o n of some jaws p r e v e n t e d a c c u r a t e m e a s u r e m e n t s of all d e n t a l r o o t s on t h e s a m e r a d i o g r a p h . Radiological m e a s u r e m e n t s were therefore verified on e x t r a c t e d teeth. Table 3 shows t h a t in t h e D i l a n t i n group t h e r o o t s of t h i r d molars were s h o r t e r a n d t h a t d r i e d M a weighed less t h a n in t h e controls. M e a s u r e m e n t s on M 1 a n d M 2 showed changes in t h e s a m e direction, b u t t h e difference b e t w e e n group m e a n s failed to r e a c h t h e 5 % significance level. F o r M a ash was 77 % of d r y weight in b o t h groups. Table 4 shows t h a t t h e e x t r a c t e d whole m a n d i b u l a r incisors from t h e D i l a n t i n g r o u p were significantly smaller t h a n those from controls ( P < 0 . 0 1 for weight a n d < 0 . 0 2 d e h y d r a t e d vol). L i n e a r m e a s u r e m e n t of r a n d o m pairs of t e e t h i n v a r i a b l y showed t h a t t h e t e e t h from e x p e r i m e n t a l animals were s h o r t e r a n d t h i n n e r a n d f r e q u e n t l y less p i g m e n t e d t h a n those of controls. H o w e v e r , t h e c o m p o s i t i o n of incisors was similar in b o t h groups (Table 4).
Fig. 4A and B. Demineralized, longitudinal section of vertebra # 13. (A) Control: joint space, joint surface, epiphyseal cartilage and subjacent normal osteogenesis for rat vertebrae; (B) Dilantin: epiphyseal cartilage and subjacent disturbed osteogenesis. The increased width of the hypertrophic zone and delayed calcification and bone remodeling result in the greater width of the whole region; because of this the joint area cannot be included in the field as it is in the control animal. Haernatoxylin and eosin, x 100 Fig. 5A and B. Demineralized, transverse section of vertebra # 8. An internal buttress is visible. A greater amount of residual calcified material is visible in the Dilantin (B) than in the Control (A). This has resulted in coarser trabeculae with larger intervening spaces. Haematoxylin and eosin, x 100 17 Calcif.Tiss. Res., Vol. 17
242
P . H . Staple and W. A. Miller
Table 3. Root length and dehydrated weight of stored and fixed third molar teeth of young adult male Charles l~iver rats receiving sodium Dilantin or vehicle. M e a n • S.E.M. number of animals in parentheses. P < 0.05 for values underlined Control (vehicle, i.p.)
Sodium Dilantin (100 mg/kg, i.p.)
Mandibular molars Length ant. buccal root (arb. units)a Length ant. lingual root Length post. root Dehydrated weight (mg)
16.8 ~-0.33 14.9 -4-0.29 16.8 :~0.41 7.53 ~- 0.08
(12) (11) (13) (13)
16.3 • (12) 14.9 • (12) 15.3 :j 0.40(13) 7.34 • 0.14 (13)
16.1 • 13.8 ~-0.25 16.2 • 6.15•
(13) (12) (13) (12)
15.0 • (9) 13.5 =]=0.46 (8) 15.0 • (9) 5.64-4-0.13 (9)
Maxillary molars Length ant. buccal root Length ant. palatal root Length post palatal root Dehydrated weight (mg) a (1 unit ~ approx. 0.1 mm). Table4. Density and composition (mg/mm a dehydrated volume) of stored and fixed mandibular incisors of young adult male Charles River rats receiving sodium Dilantin or vehicle. Mean • S.E.M. number of animals in parenthesis Controls (vehicle, i.p.)
Sodium Dilantin (100 mg/kg, i.p.)
Mandibular incisor Dehydrated weight (mg) Dehydrated volume (mm 3) Dehydrated density Ash Volatile inorg. -~ org. fraction
49.99 17.22 2.907 2.32 0.57
~:0.74 ~0.35 :~ 0.028 i0.05 •
(12) (12) (12) (12) (12)
44.88 :[=1.00 15.58 -4-0.49 2.895 • 0.049 2.28 -4-0.05 0.62 =E0.02
(13) (13) (13) (13) (13)
Contralateral mandibular incisor, basal 1/3 Dehydrated density Ash Volatile inorganic A-organic fraction
2.385 ~ 0.049 (13) 1.59 =]=0.05 (13) 0.79 :L0.02 (13)
2.282 =t=0.074 (13) 1.47 • (13) 0,82 • (13)
The m e a n l e n g t h of t h e g r e a t e s t chord of t h e labial contour of t h e m a x i l l a r y incisors (Schour a n d v a n D y k e , 1932) in r a d i o g r a p h s was 12.01 ~:0.08 m m for D i l a n t i n c o m p a r e d w i t h 12.21 -L 0.10 m m for controls ( P < 0.2 > 0.05). Observations on b a n d s in t h e c e m e n t u m are described in t h e section on h a m s t e r teeth.
Relationship between Rat Body Weight and Molar Tooth Size T h e r e was no significant correlation b e t w e e n b o d y weight a n d t h e s u m of t h e d r y weights of t h e m a n d i b u l a r m o l a r t e e t h for e i t h e r group of animals. A t best, in t h e D i l a n t i n group r = + 0.486 (t = 1.67 for dI 9, P > 0.1). Similarly, t h e r e was no correlation b e t w e e n b o d y weight a n d e i t h e r M 1 or M 3 r o o t length.
Diphenylhydantoin Effects on Bones and Teeth
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Fig. 6. Photomicrographs of distal aspect of hamster maxillary incisors in situ in dried skull preparations showing series of elevations and depressions in cementum believed to represent successive attachments of the periodontal ligament. Above: control; below: sodium Dilantin. Scale = 1 mm. The number is the average distance (in arbitrary units) between depressions (see Methods), • 9 Hamster Teeth
Measurement of hamster maxillary molar tooth root length on radiographs was inaccurate because the angulation of the roots gave distorted and overlapping images. Except Ms, the disposition of cementum made dental extraction impossible without root fracture. A comparison of the relative areas of mandibular incisors in radiographs revealed no difference ( P > 0 . 0 5 ) between Dilantin ( n = 8 ) and control groups (n = 7 ) . Similarly there were no differences between groups respecting mean dry weight, length of distal root, and ash % for 10 extracted Ma. Maxillary incisors differed in the two groups: the average distance between transverse depressions in the cementum on the distal aspect of these teeth in situ (Fig. 6) was 3 . 5 0 • arbitary units for 9 control animals, compared with 3.16 ~: 0.11 for 10 animals in the Dilantin group (P < 0.05). I n rats, several specimens had incisors with bands in the cementum like those in hamsters, but the number of teeth with well defined bands was insufficient for quantitative comparison.
Discussion The daily i.p. administration of 100 mg/kg sodium Dilantin for 26-27 days to male Charles River rats, aged 34 days at the beginning of the experiment, elicited acute neurotoxic symptoms, retarded body weight increase, and altered the development of both the caudal skeleton and the dentition. Another experiment (unpublished), imposing similar treatment for 13 days on a small number of male Long-Evans rats aged about 50 days at the beginning of the experiment, elicited the same neurotoxic symptoms and slightly retarded body weight increase without altering the size of the mandible or the development of the dentition. Probably, these differences reflect differing exposures to 17"
244
P.H. Staple and W. A. Miller
the drug relative to the developmental stage of the dentition and skeleton (Bamne et al., 1957, 1958; Scott and Symons, 1967). This hypothesis is supported by the data on the root length of molar teeth, which show that Dilantin had the greatest effect on third molars, i.e. the teeth with least completed root development when the experiment began. Similarly, the decreased size of incisors in Dilantin group reflects decreased tooth formation by active tissues in the apical region during the period of drug administration. A significant effect of Dilantin on the development of hamster M8 would not be expected because root formation was nearly complete at the beginning of the experiment (Scott and Symons, 1967). Evidently, the 25 mg/kg dose of Dilantin was not below the threshold response of dental tissues, as shown by the periodontal displasia illustrated in Fig. 6. The banding pattern illustrated in this figure is created by an alternating series of transverse elevations and depressions in the cementum, which we believe mark successive attachments of the periodontal ligament during continuous tooth eruption. The altered spacing of these bands in the Dilantin group then indicates a disturbance of the normal attachment and/or tooth eruption. Because this was a retrospective study, data concerning the effect of Dilantin on dental development are incomplete. In the case of the rats, the period of drug administration (26-27 days) was less than half that required for complete replacement of the incisor. A more definitive experiment would require a sample of younger animals and extension of the period of Dilantin administration to cover all of dental development after birth (newborn rats can tolerate biweekly intraperitoneal injections of Dilantin and are not rejected by nursing mothers, provided gloves are worn during handling and any trace of blood at the site of injection is removed). Gabler (1968) suggested that neurotoxic rats receiving sodium Dilantin (100 mg/kg) failed to gain weight normally because they were unable to acquire and eat the same amount of food as controls. In our experiment, food consumption of the Dilantin and control rats was not dissimilar as assessed by daily visual comparisons of food remaining in the containers of animals housed singly. Recently, Kraft et al. {1974) demonstrated by pair feeding that growth retardation in rats receiving Dilantin at 40 mg/kg body weight was not due to reduced food uptake. Previously, Clarke et al. (1971) demonstrated that Dilantin did not affect D-xylose absorption from the rat intestine. Although dietary deficiencies can alter dental morphology in the rat (Paynter and Grainger, 1956), we are unaware of observations indicating that proliferation of Hertwig's epithelial sheath, which controls dental root development, is affected by restriction of an otherwise nutritionally adequate diet. Also we found no correlation between final body weight of rats and dry weight of molar teeth or length of molar roots. Moreover, despite the reduced weight of rat mandibular incisors in the Dilantin group (Table 4), the composition of whole teeth or that of the contralateral basal third was similar in Dilantin and control groups ( P ~ 0 05) Validity of the analyses is established by the data showing the immaturity of the basal portion. Technical difficulties prevented analyses of molar teeth. The relationship between individual body weight and size of caudal vertebrae could not be determined because amputated tails were identified only by treatment group, not by individual animal numbers.
Diphenylhydantoin Effects on Bones and Teeth
245
Our introduction referred to reports of functional deficiency of vitamin D in patients receiving long-term Dilantin therapy. Since this vitamin stimulates body weight growth apart from its antirachitic effect (Wasserman and Corradino, 1973), the question arises whether the growth retardation of rats in our experiments resulted from vitamin D deficiency. Because this vitamin can abolish growth retardation of rats treated with Dilantin (Kraft et al., 1974), an affirmative answer to this question would be plausible. However, since Dilantin interferes with intestinal calcium absorption more directly and independently of disturbance of vitamin D metabolism (Kraft et al., 1974), and since cellular calcium levels regulate cell proliferation and protein synthesis (Wasserman and Corradino, 1973), Dilantin evidently may affect growth of rats by multiple actions. Accordingly, in the absence of reports describing teeth with short roots in vitamin Ddeficient animals, we must look for other actions of Dilantin which might influence skeletal growth and dental development. Release of rat pituitary adrenocorticotropin and ovulating hormone is depressed by Dilantin administration (Bonnycastle and Bradley, 1960; Dill, 1966; Quinn, 1965); perhaps this treatment also depresses release of growth hormone. A comparison of our findings with data on tail growth and dental development in hypophysectomized rats (Baume et al., 1957, 1958; Schour and van Dyke, 1932, 1934) reveals that the effect of chronic Dilantin administration to rats from the 31st-57th day of age simulates some of the effects of hypophyseetomy at 34-64 days of age. Nevertheless, a recent review (Martin, 1973) does not include diphenylhydantoin or other anticonvulsants among the agents causing pharmacologic blockade of growth hormone production or release. But since this release, at least in the rat, is calcium dependent (Milligan et al., 1972) and diphenylhydantoin interferes with calcium transport across cell membranes (Kraft et al., 1974) including brain synaptosomes (Sohn and Ferrendelli, 1973), at least there is a theoretical basis for pharmacologic blockade of growth hormone secretion by diphenylhydantoin. Collagen is the major organic constituent of intervertebral discs and ligaments associated with the intervertebral spaces seen in tail radiographs. Hence the increased width of these spaces in the experimental group (Table 2, Fig. 3), may reflect Dilantin's action on collagen metabolism (Bright, 1965; Cheng and Staple, 1972 ; Liu and Bhatnager, 1973). Reported stimulation of embryonic bone formation (Baratieri and Gagliardi, 1970) and accelerated healing of fractures in mice (Gudmundson and Lidgreen, 1973) and in rabbits (Sklans et al., 1967) may have a similar cause. Further investigation is necessary to explain the effect of Dilantin administration on the periodontium of hamsters. In accord with Clark et al. (1971), we found no evidence of osteomalacia or rickets in rats treated with Dilantin, which seems inconsistent with recent demonstrations that this treatment impairs calcium absorption and retention (Kraft et al., 1974). Shoshan and Pisanti {1971), however, have shown that low calcium intake in rats does not necessarily impair bone and tooth formation nor lower calcium concentration in bone ash. It is also pertinent that Dilantin administration did not change the composition of bones and teeth, in accord with data of Gudmundson and Lidgren (1973) indicating that in mice Dilantin failed to impair 24-hour uptake of SsSr into fracture callus.
246
P . H . Staple and W. A. Miller
H a m m e r s t r S m (1970) showed specific u p t a k e of D i l a n t i n b y developing r a t enamel. Our s t u d y is the first to suggest t h a t this u p t a k e m a y affect d e n t a l d e v e l o p m e n t . I n reference to our d a t a on the d e n t a l a t t a c h m e n t of the period o n t a l ligament, two previous clinical reports provide indirect evidence t h a t the reaction of periodontal s t r u c t u r e s to stress m a y be a b n o r m a l i n epileptic p a t i e n t s receiving a n t i c o n v u l s a n t t h e r a p y with D i l a n t i n (Cunat a n d C[ancio, 1969; Anon y m o u s , 1972). Since the bulk of the clinical evidence a p a r t from two reports of calvarial e n l a r g e m e n t ( R a t t a n , 1970; Lefebvre et al., 1972) indicates t h a t D i l a n t i n t h e r a p y m a y be associated with diminished bone d e n s i t y characteristic of rickets or osteomalacia, our findings emphasize the i m p o r t a n c e of species difference i n reactions to D i l a n t i n a d m i n i s t r a t i o n (cf Staple et al., 1967; Villareale et al., 1974). However, u n t i l there are more definitive studies, physicians a n d dentists should r e m a i n alert for signs of impaired d e n t a l d e v e l o p m e n t a n d altered periodontal response to stress in p a t i e n t s c o m m e n c i n g long t e r m D i l a n t i n t h e r a p y early i n life. Acknowledgements. We are indebted to Mona Everett, Lawrence Kearney and James Myles (supported by a Fellowship from the United Way, Grant No. PTF-22-UB-73) for skilful technical assistance. We appreciate helpful discussions with Dr. J. K. Gong and Dr. Leon Kraintz. We acknowledge the latter's support for preliminary experiments. This investigation was supported by an Institutional Grant from the American Cancer Society to the University of Alabama Medical College, by USPHS Research Grants DE-01932, 02091, FR 08330 and by an award from the Committee on Research of the University of British Columbia covering preliminary experiments carried out by P.H.S. while on sabbatical leave at the Department of Oral Biology, Faculty of Dentistry, University of British Columbia. Note added in proo1. Harris et al. (1974) have noted that epileptic patients on long-term treatment with anticonvulsants show a significant incidence of stunted dental root formation, which may reflect an effect of diphenylhydantoin on release of parathyroid hormone or inhibition of hormonal action on target tissue. Reference: Harris, M, Jenkins, M.V., Wills, M. R. : Phenytoin inhibition of parathyroid hormone induced bone resorption in vitro. Brit. J. Pharmacol. 50, 405408 (1974) References Anonymous: The secret of Sharlene's new smile: her own teeth~in "natural" dentures. Dent. Surv. 48 (10), 49 (1972) Baratieri, A., Gagliardi, V.: Experimental study of hyperplastic osteogenie activity and keratogenic effect of diphenylhydantoin sodium. J. dent. Res. 49, 691 (1970) Baume, L. J., Becks, H., Evans, H.M.: Hormonal control of ossification of the caudal vertebrae in the rat. I. Physiologic changes in females with increasing age. A. A metric and roentgenographic study. Helv. odout. Acta 1, 9-12 (1957) Baume, L. J., Becks, H., Evans, H.M.: Hormonal control of ossification of the caudal vertebrae in the rat. II. Changes in female rats at progressively longer intervals following hypophysectomy. Helv. odont. Acta 2, 12-19 (1958) Bonycastle, D. D., Bradley, A. J. : Diphenylhydantoin and the release of adrenocorticotropic hormone in the albino rat. Endocrinology 60, 355-363 (1960) Bright, N. H. : Effect of diphenylhydantoin on proline and hydroxyproline excretion in the rat. Proc. Soc. exp. Biol. (N.Y.) 120, 463-465 (1965) Cheng, P-T. H., Staple, P. H.: Effect of a dorsal dermal surgical wound on the chemical response of rat abdominal skin to chronic administration of sodium diphenylhydantoin. J. dent. Res. 51, 131-143 (1972) Chilton, N.W. : Design and analysis in dental and oral research, p. 94-97. Philadelphia and Toronto: Lippincott 1967
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Clark, R. L., Kuhn, J. P., Dujovne, C. A. : Absence of rickets after chronic Dilantin administration: experimental radiological observations in rats. Invest. Radiol. 6, 152-154 (1971) Cunat, J. J., Ciancio, S. G. : Diphenylhydantoin sodium: gingival hyperplasia and orthodontic treatment. Angle Orthodont. 39, 182-185 (1969) Dill, R. E. : Discrepancy of adrenal responses in diphenylhydantoin-treated rats. Arch. int. 1)harmacodyn. 160, 363-372 (1966) Gabler, W. L. : The effect of 5,5-diphenylhydantoin on the rat uterus and its fetuses. Arch. int. 1)harmacodyn. 175, 141-152 (1968) Genuth, S.M., Klein, L., Rabinovich, S., King, K . C . : Osteomalacia accompanying anticonvulsant therapy. J. clin. Endocr. 35, 378-386 (1972) Gong, J. K. : Volumetric composition of the monkey skeleton. Anat. Rec. 172, 543-550 (1972) Gong, J. K., Arnold, J. S., Cohn, S . H . : The density of organic volatile and non-volatile inorganic components of bone. Anat. Rec. 149, 319-324 (1964) Gudmundson, C., Lidgren, L. : Does diphenylhydantoin accelerate healing of fractures in mice ? Acta orthop, scan& 44, 640-649 (1973) Hammerstr5m, L.: Specific uptake of some drugs in ameloblasts and developing enamel. Acta odont, scand. 28, 187-195 (1970) Kraft, D., Herrath, D. yon, Schaeffer, K. : Retarded growth of rats by anticonvulsant drugs. Epilepsia (Boston) 15, 8 1 ~ 0 (1974) Kraintz, L., Kraintz, F.W., Ignacz, M., Staple, 1). H.: Dilantin | induced hypocalcemia in the rat. J. dent. Res. 52 (special issue) 95, I A D R Abstracts 1973, No. 146 (1973) Lefebvre, E. G., Haining, R. G., Labbe, R. F. : Coarse facies, calvarial thickening and hyperphosphatasia associated with long-term anticonvulsant therapy. New Engl. J. Med. 286, 1301-1302 (1972) Liu, T. Z., Bhatnager, R. S.: Inhibition of protocollagen proline hydroxylase by Dilantin. 1)roc. Soc. exp. Biol. (N.Y.) 142, 253-255 (1973) Martin, J. B. : Medical Progress: neural regulation of growth hormone secretion. New Engl. J. Med. 288, 1384-1393 (1973) Milligan, J . V . , Kraicer, J., Fawcett, C. 1)., Illner, 1). : Purified growth hormone releasing factor increases 4~Ca uptake into pituitary cells. Canad. J. Physiol. 1)harmacol. 50,613-617 (1972) 1)aynter, K. J., Grainger, R. M. : The relation of nutrition to the morphology and size of rat molar teeth. J. Canad. dent. Ass. 22, 519-531 (1956) Quinn, D. L. : Influence of diphenylhydantoin on spontaneous release of ovulating hormone in the adult rat. 1)roc. Soc. exp. Biol. (N.Y.) 119, 982-985 (1965) Rattan, K. R.: Calvarial thickening after Dilantin medication. Amer. J. Roentgenol. 119, 101-105 (1970) Schour, I., Dyke, H. B. van: Changes in the teeth following hypophysectomy. I. Changes in the incisor of the white rat. Amer. J. Anat. 50, 397-433 (1932) Schour, J., Dyke, H. B. van: Changes in teeth following hypophysectomy. II. Changes in the molar of the white rat. J. dent. Res. 14, 69-90 (1934) Scott, J. H., Symons, :N. B. B. : Introduction to dental anatomy, 5th ed., p. 398. Edinburgh and London: Livingstone 1967 Shoshan, S., Pisanti, S. : The metabolic effect of low calcium intake on collagen of bones and dental structures in the rat. Arch. oral Biol. 16, 791-800 (1971) Sklans, S., Taylor, R. G., Shklar, G.: Effect of diphenylhydantoin sodium on healing of experimentally produced fractures in rabbit mandibles. J. oral Surg. Anesth. 25, 310-318 (1967) Sohn, R. S., Ferrendelli, J. A. : Inhibition of Ca ++ transport into rat brain synaptosomes by by diphenylbydantoin (DPH). J. 1)harmacol. exp. Ther. 185, 272-275 (1973) Staple, 1). H., Houck, J. C., Nutter, D. J. : Absence of characteristic reactions in the periodontium and other peripheral tissues of hamsters showing neurophysiological response to chronic administration of 5,5-diphenylhydantoin (Dilantin | ). J. oral Therap. 1)harmacol. 3, 251-261 (1967) Staple, P. H., Miller, W. A. : Altered bone and tooth development in rats receiving diphenylhydantoin. J. dent. Res. 52 (special issue), 113, I A D R Abstracts No. 219, (1973a)
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Staple, P. H., Miller, W. A. : Effects of chronic administration of sodium diphenylhydantoin ("Dilantin") on teeth and vertebrae of rodents. J. dent. Res. 52, 622, IADR Regional Meeting abs. (1973b) Staple, P.H., Nutter, D . J . : Antagonism of pentobarbitone anaesthesia in hamsters by 5,5-diphenylhydantoin modified by salicylate. Nature (Lond.) 194, 944-945 (1962) Varkey, K., Raman, P. T., Bhaktaviziam, A., Taori, G. M. : Osteomalacia due to phenytoin sodium--A case report with review of literature. J. neurol. Sci. 19, 287-295 (1973) Villareale, M., Gould, L. V., Wasserman, R. H., Barr, A., Chiroff, R. T., Bergston, W. I-I.: Diphenylhydantoin: Effects on calcium metabolism in the chick. Science 183, 671-673 (1974) Wasserman, R. H., Corradino, R. A. : Vitamin D, calcium and protein syntehsis. Vitam. and Horm. 31, 43-103 (1973) Zugibe, F. T., Fink, M. L. : A new ion association technique for demonstrating polyanions in tissue sections. J. Histochem. Cytochem. 14, 147-152 (1956)
Effects of Chronic Administration of Sodium Diphenylhydantoin ('Dilantin') on Bones and Teeth of the Rat and Hamster: a Preliminary Study P. H. S t a p l e a n d W i l l i a m A. Miller Department of Oral Biology, School of Dentistry, State University of New York, Buffalo, :New York Received July 8, accepted December 8, 1974 Male Charles River rats, 31-days-old, received i.p. injection of sodium diphenylhydantoin (DPH), 100 mg/kg in 0.9% NaC1, once daily for 26-27 days before death. Male Syrian hamsters, 40-days-old, received similar injections of DPH, 25 mg/kg for 46 days, no treatment for 39 days, then DPtt for a further 17 days before sacrifice. All rats receiving DPH gained less weight than the controls, and more than 50% displayed acute neurotexic reactions to the drug; hamsters were not so affected. Morphology and composition of caudal vertebrae, teeth, and jaws from control and DPH-rats were compared on the basis of measurements on radiographs and gross specimens, histological investigation, and determination of % dry volumes of ash, volatile inorganic component, lipid, and organic matrix. DPH-vertebrae were smaller and showed impaired osteogenesis, but chondrogenesis was similar to controls. Overall tail length was similar in both animal groups because caudal intervertebral spaces were wider in DPH-rats, compensating for reduced longitudinal growth of corresponding vertebrae. Incisors were smaller and third molar roots shorter in DPH-rats. In DPH-hamsters the attachment of the periodontal ligament to maxillary incisors was deranged. DPH administration did not change the composition of rat bone or teeth. Densities of dry bones and teeth were in accord with their composition. Possible modes of action of DPH are discussed. Species differences in response of mineralized tissues to DPtt administration are emphasized in relation to reports of rickets and osteomalacia in patients on long term DPH therapy. Key words: Diphenylhydantoin - - Growth - - Skeleton - - Dentition. Introduction Bone f o r m a t i o n a n d calcium m e t a b o l i s m m a y be d i s t u r b e d in p a t i e n t s on long t e r m a n t i c o n v u l s a n t t h e r a p y ( G e n u t h et al., 1972; V a r k e y et al., 1973); b u t w h e t h e r this d i s t u r b a n c e is m e d i a t e d b y a l t e r a t i o n of v i t a m i n D m e t a b o l i s m or n o t is controversial ( K r a i n t z et al., 1973; Villareale et al., 1974). 5,Tone of these r e p o r t s m e n t i o n d e n t a l a b n o r m a l i t i e s , n o r do t h e y suggest t h a t t h e j a w bones m i g h t also be affected. Accordingly, we e x a m i n e d bones a n d t e e t h of r a t s a n d h a m s t e r s p r e s e r v e d from p r e v i o u s e x p e r i m e n t s (Staple et al., 1967 ; Cheng a n d Staple, 1972) for evidence t h a t chronic a d m i n i s t r a t i o n of d i p h e n y l h y d a n t o i n sodium ("Dilant i n " ) affects n o t only t h e skeleton b u t also t h e t e e t h a n d t h e i r s u p p o r t i n g structures. P r e l i m i n a r y a c c o u n t s of this i n v e s t i g a t i o n h a v e a l r e a d y b e e n p r e s e n t e d (Staple a n d Miller, 1973a, b).
For reprints: Dr. P. H. Staple, Dept. of Oral Biology, State University of New York, 4510 Main Street, Buffalo, :New York 14226, U.S.A.
236
P . H . Staple a n d W. A. Miller Materials and Methods
Animals, Drug Administration. The handling of male Syrian hamsters (Staple et at., 1967) a n d Charles River rats (Cheng and Staple, 1972) has been described. Essential details of these experiments, which provided bones and teeth for the present investigation, are as follows: Ten 40-day-old hamsters received daily intraperitoneal (tip.) injections of sodium Dilantin (25 mg/kg calculated from weekly individual body weights), for 46 days, no t r e a t m e n t for 39 days, then sodium Dilantin for a further 17 days before death. Ten controls were injected with vehicle. Thirteen 34-day-old Charles River rats received similar injections of sodium Dilantin (100mg/kg calculated from the daily mean body weight), for 26-27 days before death. Thirteen controls received vehicle. Rat Tails and Caudal Vertebrae. Tails from 7 treated and 7 control animals h a d been a m p u t a t e d close to the anal fold a n d stored at --20 ~. Six years later, a t room temperature, they were weighed, measured, and radiographed using 70 kV, 9 mA, a t 75 cm and K o d a k Professional Fine Grain Film. Subsequently, they were immersed overnight in 10% formalin, dried and stored. Caudal vertebrae were dissected out and either subjected to gross compositional analysis on a volumetric basis (Gong et al., 1964; Gong, 1972) or prepared for histologic examination b y routine methods. A second tail radiograph was made to check the location of vertebrae removed. Lengths of selected vertebrae and widths of intervertebral spaces were determined on the initial radiographs viewed 10 x . The microscope was equipped with a filar micrometer eyepiece reading to 0.001 ram. Overall tail length was also estimated b y measuring the length of a thread conformed to the radiographic image of the 4 t h - 2 7 t h (terminal) caudal vertebrae. Rat Teeth and Mandibular Bone. Skulls a n d disartieulated mandibles h a d been stored a t --80 ~ Three years later, they were transferred to modified Newcomer's fixative (Zugibe a n d Fink, 1966) a t --30 ~ for freeze substitution, then stored in 2-propanol a t room temperature. These bones were radiographed to display the dental roots with minimal distortion. After radiography, the t e e t h were extracted after briefly boiling the bones in dilute NH~OH solution or t a p water. Molar root length was determined on radiographs and directly on extracted teeth, b o t h viewed under 10 X, using a stereomicroscope equipped with a n eyepiece disc micrometer. The arbitrary unit employed was approximately 0.1 ram. The dehydrated weight of molar teeth was determined after incubation overnight a t 110% Maxillary third molars were ashed at 800 ~ for 48 h. The composition of an incisor and of mandibular bone (5-15 rag) from t h e angular process from each animal was determined on a volumetric basis as described for caudal vertebrae b u t using a n electrobalance. The basal third of the contralateral mandibular incisor was analysed similarly. Hamster Teeth. Cranial portions containing the maxillary dentition had been defleshed b y Dermestid beetles, washed in dilute ammonia and air dried before storage at room temperature for 10 years. Each preparation was bisected a n d radiographcd. Radiographs were projected a t 6 X onto paper a n d the relative areas of right or left maxillary incisors were determined b y the paper weight method. A series of transverse bands in cementum on the distal surface of the maxillary incisor became visible when the maxilla was suitably oriented a n d viewed b y oblique incident illumination using a steromieroscope a t 10 • Beginning near the ineisal edge, the distance between the first 10 bands was measured with a n eyepiece disc micrometer. The length of the distal root of extracted third maxillary molars was measured, as described for rats. These teeth were t h e n ashed at 650 ~ for 102 h, or a t 800 ~ for 48 h. All data were subjected to a n analysis of variance to determine their significance.
Results
Acute IVeurotoxic Response to Sodium Dilantin F i g u r e 1 s h o w s t h a t a d m i n i s t r a t i o n of 100 m g / k g s o d i u m D i l a n t i n c a u s e d a c u t e n e u r o t o x i c r e a c t i o n s i n a t l e a s t 5 0 % of t h e r a t s . C o n t r o l s b e h a v e d n o r m a l l y .
Diphenylhydantoin Effects on Bones and Teeth
237
260~24O 220i E
Controls
200-t-
180o
rn
Dilantin Sodium
160140120-I00-.:
eo~ 25
50
55
40
45
50
55
60
DAYS
Fig. 1. Male Charles River rats. Above: fractional daily incidence of neurotoxic reactions in treated rats occurring within 20 min of intraperitoneal (i.p,) injection of sodium Dilantin, 100 mg/kg body weight, suspended in 0.9% NaC1. A value of 0.6 indicates that 8 of 13 rats displayed neurotoxicity. Below: days of age and mean body weight of 13 rats receiving sodium Dilantin, 100 mg/kg, as above, and 13 rats receiving saline vehicle, i.p. The arrow indicates commencement of Dilantin or saline injections
O v e r t n e u r o t o x i c i t y was a b s e n t in h a m s t e r s receiving 25 m g / k g s o d i u m Dilantin, b u t these a n i m a l s r e s p o n d e d a b n o r m a l l y to p e n t o b a r b i t a l a n e s t h e s i a (Staple a n d N u t t e r , 1962). A n i m a l Growth
R a t s receiving sodium D i l a n t i n failed to grow at t h e s a m e r a t e as controls (Fig. 1). A t d e a t h a f t e r 26 or 27 d a y s t r e a t m e n t , t h e m e a n b o d y weight of t h e t r e a t e d group was 232.1 :h 5.5 (S.E.M.) g c o m p a r e d with 270.0 :[: 7.1 g for controls ( P < 0.01). I n i t i a l l y , some h a m s t e r s receiving sodium D i l a n t i n failed to gain weight as r a p i d l y as controls. W e i g h t gain of these a n i m a l s d i d n o t a c c e l e r a t e d u r i n g d r u g w i t h d r a w a l n o r level off w h e n D i l a n t i n was r e a d m i n i s t e r e d . W h e n a n i m a l s were sacrificed a t 142 days, t h e m e a n b o d y weight of t h e D i l a n t i n group was 150.6 4- 6.8 g (10) c o m p a r e d with 157.6 ~ 3.3 (10) for controls ( P > 0.05).
238
P.H. Staple and W. A. Miller
Table 1. Measurements of lengths on radiographs of caudal vertebrae in stored amputated tails of young adult male Charles River rats receiving sodium Dilantin or vehicle Group of 7 Animals Control (vehicle, i.p.) Sodium Dilantin (100 mg/kg, i.p.)
Mean length (mm) of caudal vertebrate number 5
9
13
17
21
25
Group grand mean length (mm of caudal vertebra
9.065
8.712
8.150
6.929
5.134
3.016
6.834
8.715
8.444
7.872
6.668
5.020
2.960
6.614
Rat Tails and Caudal Vertebrae Length. The mean length of thawed stored tails from 7 treated animals was 15.8• (S.E.M.) cm compared with 16.6~0.22 cm for 7 controls. Corresponding tail weights were 2.6 • 0.13 and 3.3 ~ 0.16 g. These differences (P ~0.05) were found to be spurious following comparison of the tail radiographs with those of previous authors (Baume et al., 1957), which revealed non-uniform tail amputations. Re-determination of tail lengths, measuring between the distal ends of the 4th and 27th (terminal) vertebrate on the radiographs, showed no difference between experimental and control groups (P ~ 0.05). The components of tail length were further investigated by measuring the lengths of vertebrae ~ 5, 9, 13, 17, 21, and 25, and the widths of the corresponding intervertebral spaces, excepting # 25. Table 1 shows that the mean length of selected caudal vertebrae in the Dilantin group was less than in the control group. The complete data also show that in the control group vertebral length progressively declined from # 5-21, whereas in the case of 2 of 7 treated animals vertebra # 9 was longer than # 5. A 3-way analysis of variance (Chilton, 1967) showed that: (1) F = b e t w e e n groups mean square/between animals (within groups) mean square = 1.24 for df 1, 12 ; P ~ 0.05. (2) F = vertebrae • group interaction mean square/animals • vertebrae interaction (within groups) mean square = 1376.6 for df 5, 60; P < 0 . 0 0 1 . This indicates that although the difference between group grand means is not significant, the groups differ significantly when compared on the basis of the mean values for corresponding individual vertebrae in the series. Table 2 shows that: (1) I n both control and treated groups the mean width of caudal intervertebral spaces progressively declined from ~ 4-22. (2) Beginning at intervertebral space ~ 12-13, mean space width in the Dilantin group was greater than in controls. Three-way analysis of variance (a) on the complete data, (b) for data commencing with intervertebral space # 12-13, showed no significant differences between group grand means (P ~ 0.05), nor between means for each intervertebral space considered separately. However, when groups were compared commencing with intervertebral space # 12-13, F = intervertebral space • group interaction mean square/animals • intervertebral space interaction (within groups) mean square = 4.79 for df 5.60; P = 0.001. This indicates a significant difference between the Dilantin and control group intervertebral space widths in the segment of tail comprising intervertebral spaces # 12-22.
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Table 2. Measurements on radiographs of widths of caudal intervertebral spaces in stored amputated tails of young male adult Charles River rats receiving sodium Dilantin or vehicle Group of 7 Animals
Control (vehicle, i.p.)
Mean width (mm) of caudal intervertebral space number 4-5
5 6
8-9
9-10
12-13
13-14
16-17
17-18
20-21
21-22
Group grand mean width of caudal intervertebral space a (a)
(b)
0.664 0.565 0.367 0.327 0.226
0.208
0.198
0.181
0.145
0.126
0.301
0.181
Sodium Dilantin 0.626 0.602 0.360 0.324 0.309 (100 mg/kg, tip.)
0.239
0.228
0.204
0.195
0.173
0.325
0.225
a (a) For whole series # 4 - 5 .... 21-22, (b) # 12-13 .... 21 22 only.
Composition. Caudal v e r t e b r a e # 6 a n d 7 were t a k e n for c o m p o s i t i o n a l analyses. F o r # 6, t h e m e a n d e h y d r a t e d weight was 43.42 ~ 2.50 (S.E.M.) m g for D i l a n t i n compared with 58.14• for control a n d t h e m e a n v o l u m e was 19.66 :~ 1.44 m m a c o m p a r e d w i t h 27.37 • 1.44 m m 3 ( P = 0.01, n = 6) ; corresponding mear~ densities were 2.240 • 0.161 rag/ram a a n d 2.137 =t=0.097 m g / m m a ( P ~ 0.05). F o r # 7 , m e a n d e h y d r a t e d weight a n d volume in t h e D i l a n t i n group were r e s p e c t i v e l y 44.87 • 3.84 m g a n d 20.96 • 2.43 m m 8 c o m p a r e d with 50.04 ~ 2.66 m g a n d 24.95 • 1.10 m m a in t h e control g r o u p ( P > 0.05 for n : 3 ) ; densities were 2.156 ~- 0.068 m g / m m 3 a n d 2.004 =~ 0.026 m g / m m a r e s p e c t i v e l y . On a v o l u m e p e r cent basis, t h e r e were no differences b e t w e e n groups r e s p e c t i n g m e a n values for ash, volatile inorganic fraction, lipid, a n d organic m a t r i x ( P > 0.05). Because of technical difficulties, lipid a n d organic m a t r i x fractions were d e t e r m i n e d o n l y on 4/6 a v a i l a b l e v e r t e b r a e # 6 from each group. Consequently, t h e differences b e t w e e n m e a n values for D i l a n t i n a n d control, r e s p e c t i v e l y 29.2• a n d 34.4:]=6.1 p e r cent, a n d 3 8 . 9 : L 3 . 7 a n d 25.6-]-5.4 p e r cent, failed to r e a c h significance a t t h e 5 % level. Anatomy and Histology. The c o n v o l u t e d contour of tail v e r t e b r a e was m o r e p r o m i n e n t in t h e p r o x i m a l v e r t e b r a e , giving a f l u t e d a p p e a r a n c e along t h e long axis of each bone. T h e fluting is analogous to the t r a n s v e r s e a n d a r t i c u l a r processes of t h e dorsal v e r t e b r a e . T h e r e was also i n t e r n a l buttressing. Because of t h e local h e t e r o g e n e i t y of tissues, g r e a t care was exercised to c o m p a r e c o m p a r a b l e regions. Most of t h e following descriptions a p p l y to t h e midline, where c o n t o u r i n g of t h e a r t i c u l a t i n g surfaces was minimal. A l t h o u g h cellular f i x a t i o n was poor, t h a t of t h e calcified tissues was sufficiently good to give reliable o b s e r v a t i o n s on t h e i r structure. The h i s t o l o g y of v e r t e b r a e # 12-14 showed t h a t t h e j o i n t space was e n l a r g e d in those a n i m a l s which h a d received Dilantin. I n controls, t h e s e c o n d a r y cartilage h a d v i r t u a l l y d i s a p p e a r e d ; however, in t h e e x p e r i m e n t a l a n i m a l s t h e r e was residual s e c o n d a r y cartilage d i s t r i b u t e d i r r e g u l a r l y b o t h c e n t r a l l y a n d p e r i p h e r a l l y a n d t h e d i s t a n c e b e t w e e n joint surface a n d t h e e p i p h y s e a l cartilage was m u c h g r e a t e r (Fig. 2 A a n d B).
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P . H . Staple and W. A. Miller
Fig. 2A and B. Demineralized, longitudinal section of epiphyseal region of vertebra @ 13. Remains of the intervertebral disc are visible (displaced in B). Greater irregularity of calcification is visible in the experimental animal (B) along with greater amounts of residual cartilage, particularly at the joint surface. Haematoxylin and van Gieson, X 40 Fig. 3A and B. Radiographs of tail vertebrae @ 12-14: (A) Control; (B) Dilantin. In B, the wider joint spaces and epiphyseal spaces are clearly visible, as well as interrupted growth lines (arrows). Both vertebrae show longitudinal fluting
Diphenylhydantoin Effects on Bones and Teeth
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E n l a r g e d c o n t a c t r a d i o g r a p h s showed these f e a t u r e s as well as t h e v a r i a t i o n s in v e r t e b r a l size a n d a difference in t r a b e c u l a r coirfiguration (Fig. 3 A a n d B). Some of t h e D i l a n t i n v e r t e b r a e showed i n t e r r u p t e d g r o w t h lines, which could n o t be r e l a t e d to t h e e x p e r i m e n t a l procedures. I n t h e e p i p h y s e a l cartilage t h e p r o l i f e r a t i n g zone was essentially similar in t h e e x p e r i m e n t a l animals a n d controls (Figs. 2 a n d 4 A a n d B), e x c e p t t h a t in some areas c h o n d r o b l a s t clones were more sparse a n d often m o r e irregular in a r r a n g e m e n t in t h e e x p e r i m e n t a l group. I n D i l a n t i n - t r e a t e d animals, t h e h y p e r t r o p h i c a n d calcifying zones were wider with more residual calcified cartilage in a f r e q u e n t l y wider s u b j a c e n t zone of basophilic w o v e n bone. The bone of t h e shaft was t h i n n e r w i t h m o r e irregular lamellae. I n b o t h e x p e r i m e n t a l a n d control a n i m a l s a r e c e n t m a j o r i n t e r n a l r e m o d e l l i n g was evident. L o n g i t u d i n a l sections of v e r t e b r a e # 8 a n d 9, a d j a c e n t to those whose composition was d e t e r m i n e d , showed t h a t t h e y were similar in s t r u c t u r e to t h e m o r e d i s t a l v e r t e b r a e a l t h o u g h t h e i n t e r v e r t e b r a l spaces were" n o t different (Table 2). I n t r a n s v e r s e sections of these v e r t e b r a e , a r o u n d t h e i n t e r n a l buttressing, i n d i v i d u a l t r a b e c u l a e were coarser with larger i n t e r v e n i n g spaces (Fig. 5 A a n d B).
Rat Mandible D i l a n t i n a d m i n i s t r a t i o n d i d n o t a l t e r t h e d r y d e n s i t y nor v o l u m e t r i c composit i o n of bone from t h e m a n d i b u l a r a n g u l a r process.
Rat Teeth The a n g u l a t i o n of some jaws p r e v e n t e d a c c u r a t e m e a s u r e m e n t s of all d e n t a l r o o t s on t h e s a m e r a d i o g r a p h . Radiological m e a s u r e m e n t s were therefore verified on e x t r a c t e d teeth. Table 3 shows t h a t in t h e D i l a n t i n group t h e r o o t s of t h i r d molars were s h o r t e r a n d t h a t d r i e d M a weighed less t h a n in t h e controls. M e a s u r e m e n t s on M 1 a n d M 2 showed changes in t h e s a m e direction, b u t t h e difference b e t w e e n group m e a n s failed to r e a c h t h e 5 % significance level. F o r M a ash was 77 % of d r y weight in b o t h groups. Table 4 shows t h a t t h e e x t r a c t e d whole m a n d i b u l a r incisors from t h e D i l a n t i n g r o u p were significantly smaller t h a n those from controls ( P < 0 . 0 1 for weight a n d < 0 . 0 2 d e h y d r a t e d vol). L i n e a r m e a s u r e m e n t of r a n d o m pairs of t e e t h i n v a r i a b l y showed t h a t t h e t e e t h from e x p e r i m e n t a l animals were s h o r t e r a n d t h i n n e r a n d f r e q u e n t l y less p i g m e n t e d t h a n those of controls. H o w e v e r , t h e c o m p o s i t i o n of incisors was similar in b o t h groups (Table 4).
Fig. 4A and B. Demineralized, longitudinal section of vertebra # 13. (A) Control: joint space, joint surface, epiphyseal cartilage and subjacent normal osteogenesis for rat vertebrae; (B) Dilantin: epiphyseal cartilage and subjacent disturbed osteogenesis. The increased width of the hypertrophic zone and delayed calcification and bone remodeling result in the greater width of the whole region; because of this the joint area cannot be included in the field as it is in the control animal. Haernatoxylin and eosin, x 100 Fig. 5A and B. Demineralized, transverse section of vertebra # 8. An internal buttress is visible. A greater amount of residual calcified material is visible in the Dilantin (B) than in the Control (A). This has resulted in coarser trabeculae with larger intervening spaces. Haematoxylin and eosin, x 100 17 Calcif.Tiss. Res., Vol. 17
242
P . H . Staple and W. A. Miller
Table 3. Root length and dehydrated weight of stored and fixed third molar teeth of young adult male Charles l~iver rats receiving sodium Dilantin or vehicle. M e a n • S.E.M. number of animals in parentheses. P < 0.05 for values underlined Control (vehicle, i.p.)
Sodium Dilantin (100 mg/kg, i.p.)
Mandibular molars Length ant. buccal root (arb. units)a Length ant. lingual root Length post. root Dehydrated weight (mg)
16.8 ~-0.33 14.9 -4-0.29 16.8 :~0.41 7.53 ~- 0.08
(12) (11) (13) (13)
16.3 • (12) 14.9 • (12) 15.3 :j 0.40(13) 7.34 • 0.14 (13)
16.1 • 13.8 ~-0.25 16.2 • 6.15•
(13) (12) (13) (12)
15.0 • (9) 13.5 =]=0.46 (8) 15.0 • (9) 5.64-4-0.13 (9)
Maxillary molars Length ant. buccal root Length ant. palatal root Length post palatal root Dehydrated weight (mg) a (1 unit ~ approx. 0.1 mm). Table4. Density and composition (mg/mm a dehydrated volume) of stored and fixed mandibular incisors of young adult male Charles River rats receiving sodium Dilantin or vehicle. Mean • S.E.M. number of animals in parenthesis Controls (vehicle, i.p.)
Sodium Dilantin (100 mg/kg, i.p.)
Mandibular incisor Dehydrated weight (mg) Dehydrated volume (mm 3) Dehydrated density Ash Volatile inorg. -~ org. fraction
49.99 17.22 2.907 2.32 0.57
~:0.74 ~0.35 :~ 0.028 i0.05 •
(12) (12) (12) (12) (12)
44.88 :[=1.00 15.58 -4-0.49 2.895 • 0.049 2.28 -4-0.05 0.62 =E0.02
(13) (13) (13) (13) (13)
Contralateral mandibular incisor, basal 1/3 Dehydrated density Ash Volatile inorganic A-organic fraction
2.385 ~ 0.049 (13) 1.59 =]=0.05 (13) 0.79 :L0.02 (13)
2.282 =t=0.074 (13) 1.47 • (13) 0,82 • (13)
The m e a n l e n g t h of t h e g r e a t e s t chord of t h e labial contour of t h e m a x i l l a r y incisors (Schour a n d v a n D y k e , 1932) in r a d i o g r a p h s was 12.01 ~:0.08 m m for D i l a n t i n c o m p a r e d w i t h 12.21 -L 0.10 m m for controls ( P < 0.2 > 0.05). Observations on b a n d s in t h e c e m e n t u m are described in t h e section on h a m s t e r teeth.
Relationship between Rat Body Weight and Molar Tooth Size T h e r e was no significant correlation b e t w e e n b o d y weight a n d t h e s u m of t h e d r y weights of t h e m a n d i b u l a r m o l a r t e e t h for e i t h e r group of animals. A t best, in t h e D i l a n t i n group r = + 0.486 (t = 1.67 for dI 9, P > 0.1). Similarly, t h e r e was no correlation b e t w e e n b o d y weight a n d e i t h e r M 1 or M 3 r o o t length.
Diphenylhydantoin Effects on Bones and Teeth
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Fig. 6. Photomicrographs of distal aspect of hamster maxillary incisors in situ in dried skull preparations showing series of elevations and depressions in cementum believed to represent successive attachments of the periodontal ligament. Above: control; below: sodium Dilantin. Scale = 1 mm. The number is the average distance (in arbitrary units) between depressions (see Methods), • 9 Hamster Teeth
Measurement of hamster maxillary molar tooth root length on radiographs was inaccurate because the angulation of the roots gave distorted and overlapping images. Except Ms, the disposition of cementum made dental extraction impossible without root fracture. A comparison of the relative areas of mandibular incisors in radiographs revealed no difference ( P > 0 . 0 5 ) between Dilantin ( n = 8 ) and control groups (n = 7 ) . Similarly there were no differences between groups respecting mean dry weight, length of distal root, and ash % for 10 extracted Ma. Maxillary incisors differed in the two groups: the average distance between transverse depressions in the cementum on the distal aspect of these teeth in situ (Fig. 6) was 3 . 5 0 • arbitary units for 9 control animals, compared with 3.16 ~: 0.11 for 10 animals in the Dilantin group (P < 0.05). I n rats, several specimens had incisors with bands in the cementum like those in hamsters, but the number of teeth with well defined bands was insufficient for quantitative comparison.
Discussion The daily i.p. administration of 100 mg/kg sodium Dilantin for 26-27 days to male Charles River rats, aged 34 days at the beginning of the experiment, elicited acute neurotoxic symptoms, retarded body weight increase, and altered the development of both the caudal skeleton and the dentition. Another experiment (unpublished), imposing similar treatment for 13 days on a small number of male Long-Evans rats aged about 50 days at the beginning of the experiment, elicited the same neurotoxic symptoms and slightly retarded body weight increase without altering the size of the mandible or the development of the dentition. Probably, these differences reflect differing exposures to 17"
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P.H. Staple and W. A. Miller
the drug relative to the developmental stage of the dentition and skeleton (Bamne et al., 1957, 1958; Scott and Symons, 1967). This hypothesis is supported by the data on the root length of molar teeth, which show that Dilantin had the greatest effect on third molars, i.e. the teeth with least completed root development when the experiment began. Similarly, the decreased size of incisors in Dilantin group reflects decreased tooth formation by active tissues in the apical region during the period of drug administration. A significant effect of Dilantin on the development of hamster M8 would not be expected because root formation was nearly complete at the beginning of the experiment (Scott and Symons, 1967). Evidently, the 25 mg/kg dose of Dilantin was not below the threshold response of dental tissues, as shown by the periodontal displasia illustrated in Fig. 6. The banding pattern illustrated in this figure is created by an alternating series of transverse elevations and depressions in the cementum, which we believe mark successive attachments of the periodontal ligament during continuous tooth eruption. The altered spacing of these bands in the Dilantin group then indicates a disturbance of the normal attachment and/or tooth eruption. Because this was a retrospective study, data concerning the effect of Dilantin on dental development are incomplete. In the case of the rats, the period of drug administration (26-27 days) was less than half that required for complete replacement of the incisor. A more definitive experiment would require a sample of younger animals and extension of the period of Dilantin administration to cover all of dental development after birth (newborn rats can tolerate biweekly intraperitoneal injections of Dilantin and are not rejected by nursing mothers, provided gloves are worn during handling and any trace of blood at the site of injection is removed). Gabler (1968) suggested that neurotoxic rats receiving sodium Dilantin (100 mg/kg) failed to gain weight normally because they were unable to acquire and eat the same amount of food as controls. In our experiment, food consumption of the Dilantin and control rats was not dissimilar as assessed by daily visual comparisons of food remaining in the containers of animals housed singly. Recently, Kraft et al. {1974) demonstrated by pair feeding that growth retardation in rats receiving Dilantin at 40 mg/kg body weight was not due to reduced food uptake. Previously, Clarke et al. (1971) demonstrated that Dilantin did not affect D-xylose absorption from the rat intestine. Although dietary deficiencies can alter dental morphology in the rat (Paynter and Grainger, 1956), we are unaware of observations indicating that proliferation of Hertwig's epithelial sheath, which controls dental root development, is affected by restriction of an otherwise nutritionally adequate diet. Also we found no correlation between final body weight of rats and dry weight of molar teeth or length of molar roots. Moreover, despite the reduced weight of rat mandibular incisors in the Dilantin group (Table 4), the composition of whole teeth or that of the contralateral basal third was similar in Dilantin and control groups ( P ~ 0 05) Validity of the analyses is established by the data showing the immaturity of the basal portion. Technical difficulties prevented analyses of molar teeth. The relationship between individual body weight and size of caudal vertebrae could not be determined because amputated tails were identified only by treatment group, not by individual animal numbers.
Diphenylhydantoin Effects on Bones and Teeth
245
Our introduction referred to reports of functional deficiency of vitamin D in patients receiving long-term Dilantin therapy. Since this vitamin stimulates body weight growth apart from its antirachitic effect (Wasserman and Corradino, 1973), the question arises whether the growth retardation of rats in our experiments resulted from vitamin D deficiency. Because this vitamin can abolish growth retardation of rats treated with Dilantin (Kraft et al., 1974), an affirmative answer to this question would be plausible. However, since Dilantin interferes with intestinal calcium absorption more directly and independently of disturbance of vitamin D metabolism (Kraft et al., 1974), and since cellular calcium levels regulate cell proliferation and protein synthesis (Wasserman and Corradino, 1973), Dilantin evidently may affect growth of rats by multiple actions. Accordingly, in the absence of reports describing teeth with short roots in vitamin Ddeficient animals, we must look for other actions of Dilantin which might influence skeletal growth and dental development. Release of rat pituitary adrenocorticotropin and ovulating hormone is depressed by Dilantin administration (Bonnycastle and Bradley, 1960; Dill, 1966; Quinn, 1965); perhaps this treatment also depresses release of growth hormone. A comparison of our findings with data on tail growth and dental development in hypophysectomized rats (Baume et al., 1957, 1958; Schour and van Dyke, 1932, 1934) reveals that the effect of chronic Dilantin administration to rats from the 31st-57th day of age simulates some of the effects of hypophyseetomy at 34-64 days of age. Nevertheless, a recent review (Martin, 1973) does not include diphenylhydantoin or other anticonvulsants among the agents causing pharmacologic blockade of growth hormone production or release. But since this release, at least in the rat, is calcium dependent (Milligan et al., 1972) and diphenylhydantoin interferes with calcium transport across cell membranes (Kraft et al., 1974) including brain synaptosomes (Sohn and Ferrendelli, 1973), at least there is a theoretical basis for pharmacologic blockade of growth hormone secretion by diphenylhydantoin. Collagen is the major organic constituent of intervertebral discs and ligaments associated with the intervertebral spaces seen in tail radiographs. Hence the increased width of these spaces in the experimental group (Table 2, Fig. 3), may reflect Dilantin's action on collagen metabolism (Bright, 1965; Cheng and Staple, 1972 ; Liu and Bhatnager, 1973). Reported stimulation of embryonic bone formation (Baratieri and Gagliardi, 1970) and accelerated healing of fractures in mice (Gudmundson and Lidgreen, 1973) and in rabbits (Sklans et al., 1967) may have a similar cause. Further investigation is necessary to explain the effect of Dilantin administration on the periodontium of hamsters. In accord with Clark et al. (1971), we found no evidence of osteomalacia or rickets in rats treated with Dilantin, which seems inconsistent with recent demonstrations that this treatment impairs calcium absorption and retention (Kraft et al., 1974). Shoshan and Pisanti {1971), however, have shown that low calcium intake in rats does not necessarily impair bone and tooth formation nor lower calcium concentration in bone ash. It is also pertinent that Dilantin administration did not change the composition of bones and teeth, in accord with data of Gudmundson and Lidgren (1973) indicating that in mice Dilantin failed to impair 24-hour uptake of SsSr into fracture callus.
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P . H . Staple and W. A. Miller
H a m m e r s t r S m (1970) showed specific u p t a k e of D i l a n t i n b y developing r a t enamel. Our s t u d y is the first to suggest t h a t this u p t a k e m a y affect d e n t a l d e v e l o p m e n t . I n reference to our d a t a on the d e n t a l a t t a c h m e n t of the period o n t a l ligament, two previous clinical reports provide indirect evidence t h a t the reaction of periodontal s t r u c t u r e s to stress m a y be a b n o r m a l i n epileptic p a t i e n t s receiving a n t i c o n v u l s a n t t h e r a p y with D i l a n t i n (Cunat a n d C[ancio, 1969; Anon y m o u s , 1972). Since the bulk of the clinical evidence a p a r t from two reports of calvarial e n l a r g e m e n t ( R a t t a n , 1970; Lefebvre et al., 1972) indicates t h a t D i l a n t i n t h e r a p y m a y be associated with diminished bone d e n s i t y characteristic of rickets or osteomalacia, our findings emphasize the i m p o r t a n c e of species difference i n reactions to D i l a n t i n a d m i n i s t r a t i o n (cf Staple et al., 1967; Villareale et al., 1974). However, u n t i l there are more definitive studies, physicians a n d dentists should r e m a i n alert for signs of impaired d e n t a l d e v e l o p m e n t a n d altered periodontal response to stress in p a t i e n t s c o m m e n c i n g long t e r m D i l a n t i n t h e r a p y early i n life. Acknowledgements. We are indebted to Mona Everett, Lawrence Kearney and James Myles (supported by a Fellowship from the United Way, Grant No. PTF-22-UB-73) for skilful technical assistance. We appreciate helpful discussions with Dr. J. K. Gong and Dr. Leon Kraintz. We acknowledge the latter's support for preliminary experiments. This investigation was supported by an Institutional Grant from the American Cancer Society to the University of Alabama Medical College, by USPHS Research Grants DE-01932, 02091, FR 08330 and by an award from the Committee on Research of the University of British Columbia covering preliminary experiments carried out by P.H.S. while on sabbatical leave at the Department of Oral Biology, Faculty of Dentistry, University of British Columbia. Note added in proo1. Harris et al. (1974) have noted that epileptic patients on long-term treatment with anticonvulsants show a significant incidence of stunted dental root formation, which may reflect an effect of diphenylhydantoin on release of parathyroid hormone or inhibition of hormonal action on target tissue. Reference: Harris, M, Jenkins, M.V., Wills, M. R. : Phenytoin inhibition of parathyroid hormone induced bone resorption in vitro. Brit. J. Pharmacol. 50, 405408 (1974) References Anonymous: The secret of Sharlene's new smile: her own teeth~in "natural" dentures. Dent. Surv. 48 (10), 49 (1972) Baratieri, A., Gagliardi, V.: Experimental study of hyperplastic osteogenie activity and keratogenic effect of diphenylhydantoin sodium. J. dent. Res. 49, 691 (1970) Baume, L. J., Becks, H., Evans, H.M.: Hormonal control of ossification of the caudal vertebrae in the rat. I. Physiologic changes in females with increasing age. A. A metric and roentgenographic study. Helv. odout. Acta 1, 9-12 (1957) Baume, L. J., Becks, H., Evans, H.M.: Hormonal control of ossification of the caudal vertebrae in the rat. II. Changes in female rats at progressively longer intervals following hypophysectomy. Helv. odont. Acta 2, 12-19 (1958) Bonycastle, D. D., Bradley, A. J. : Diphenylhydantoin and the release of adrenocorticotropic hormone in the albino rat. Endocrinology 60, 355-363 (1960) Bright, N. H. : Effect of diphenylhydantoin on proline and hydroxyproline excretion in the rat. Proc. Soc. exp. Biol. (N.Y.) 120, 463-465 (1965) Cheng, P-T. H., Staple, P. H.: Effect of a dorsal dermal surgical wound on the chemical response of rat abdominal skin to chronic administration of sodium diphenylhydantoin. J. dent. Res. 51, 131-143 (1972) Chilton, N.W. : Design and analysis in dental and oral research, p. 94-97. Philadelphia and Toronto: Lippincott 1967
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